This invention is directed in one aspect to compounds useful in the preparation of novel universal support media. The universal support media thus prepared are useful in the preparation of oligomeric compounds.
Support bound oligonucleotide synthesis relies on sequential addition of nucleotides to one end of a growing chain. Typically, a first nucleoside is attached to an appropriate support medium such as a glass bead support and activated phosphorus compounds (typically nucleotide phosphoramidites, also bearing appropriate protecting groups) are added stepwise to elongate the growing oligonucleotide. When the chain elongation is completed, the oligonucleotide is cleaved from its support and protecting groups are removed. Additional methods for support bound synthesis methods may be found in Caruthers U.S. Pat. Nos. 4,415,732; 4,458,066; 4,500,707; 4,668,777; 4,973,679; and 5,132,418; and Koster U.S. Pat. No. 4,725,677 and U.S. Re. Pat. No. 34,069.
In carrying out standard oligonucleotide syntheses, workers minimally need to maintain a supply of eight different nucleoside-loaded supports for DNA and RNA syntheses, each prederivatized with a separate nucleoside corresponding to the 3xe2x80x2 terminus of the desired oligomer (adenosine, guanosine, cytidine, uridine, deoxyadenosine, deoxyguanosine, deoxycytidine, and thymidine). If a modified nucleoside is desired at the 3xe2x80x2-terminus then additional prederivatized supports are required. Typically, the first nucleoside is covalently bound by a succinate or hydroquinone-O,Oxe2x80x2-diacetate linker. Furthermore, certain oligonucleotides with unusual nucleosides are available only as phosphoroamidites but not as supports.
A universal support is a support that may be used as a starting point for oligonucleotide synthesis regardless of the nucleoside species at the 3xe2x80x2 end of the sequence. A universal support has broad application and remedies the aforementioned deficiencies of standard oligonucleotide synthesis procedures because only one support is needed to carry out the oligonucleotide synthesis regardless of what base is desired at the 3xe2x80x2 end. This simplifies the synthetic strategy, reduces the number of required reagents in inventory and reduces the likelihood of errors in parallel synthesis applications.
Some researchers have employed derivatized glass supports with 2xe2x80x2(3xe2x80x2)-O-benzyoluridine 5xe2x80x2-O-succinyl so that the uridine moiety is linked to the glass via a succinate linkage [deBear et al., Nucleosides and Nucleotides 6, 821-830 (1987)]. Oligonucleotide synthesis takes place by adding nucleotide monomers to the 2xe2x80x2 or 3xe2x80x2 position of the uridine. Following the synthesis, the newly synthesized oligonucleotide is released from the glass, deprotected and cleaved from the uridinyl terminus in one reaction. Since it is cleaved from the solid support in the cleaving reaction, the uridinyl functionality is no longer available for subsequent oligonucleotide syntheses.
In a similar approach, Crea et al. prepared the dimer 5xe2x80x2-O-p-chlorophenylphospho-2xe2x80x2(3xe2x80x2)-O-acetyluridinyl-[2xe2x80x2-(3xe2x80x2)-3xe2x80x2]-5xe2x80x2-O-dimethoxytritylthymidine p-chlorophenylester and attached the dimer to cellulose via a phosphate linkage. The 5xe2x80x2 position of the thymidine is available for oligonucleotide attachment and synthesis. [Crea et al., Nucleic Acids Research 8, 2331 (1980)]. Aqueous concentrated ammonia is used to the release of the synthesized oligonucleotide from the cellulose leaving the uridine portion of the dimer attached to the cellulose. Although Crea et al. utilized the reactive vicinal groups on the uridine as the release site for the oligonucleotide from the uridine the solid support suggested in this reference is not truly a universal solid support because the 3xe2x80x2-terminal oligonucleotide is incorporated in the solid support reagent and a different support is required for oligonucleotides incorporating a different first nucleoside.
Schwartz et al. attached an adapter, 2xe2x80x2-(3xe2x80x2)-O-dimethoxytrityl-3xe2x80x2-(2xe2x80x2)-O-benzoyluridine-5xe2x80x2-O-(2-cyanoethyl-N,N-diisopropylphosphoramidite, to a thymidine derivatized polystyrene and synthesized an oligonucleotide from the O-dimethoxytrityl position of the uridine [Schwartz et al., Tetrahedron Letters, 36, 1, 27-30, 1995]. While this approach provides a universal solid support for oligonucleotide synthesis, cleavage releases both the adapter and the thymidine from the support and then the synthesized oligonucleotide from the uridine. Thus, thymidine linker must be removed as an impurity and the solid support is unavailable for subsequent reactions.
Some universal supports require cleavage under conditions supplemental to ammonium hydroxide, [Lyttle et al., Nucleic Acids Research, 1996, 24, 14, 2793-2798] making them less useful in many conventional syntheses where ammonium hydroxide is used as cleavage reagent.
The compounds, compositions and processes of the invention provide novel universal support media useful for preparing oligomeric compounds, including oligonucleotides and oligonucleotide mimetics, which may be effectively cleaved without rendering the support media unavailable for subsequent reactions.
In one embodiment, the invention is directed to compounds of Formula I: 
wherein
X is CH2, O, S or NR3;
R3 is alkyl, xe2x80x94C(xe2x95x90O)alkyl or an amino protecting group;
one of R1 and R2 is xe2x80x94(L)n-sm and the other of R1 and R2 is xe2x80x94C(xe2x95x90O)xe2x80x94R4 or xe2x80x94C(xe2x95x90S)xe2x80x94R4;
L is a linking moiety;
n is 0 or 1;
sm is a support medium;
R4 is xe2x80x94O-alkyl, xe2x80x94N(J1)J2;
J1 is H or alkyl;
J2 is alkyl or a nitrogen-protecting group;
or J1 and J2 together with the nitrogen atom they are attached to form a ring structure; and
Z1 and Z2 are orthogonal hydroxyl protecting groups.
Preferably, X is O, S or NR3. Preferably, R3 is alkyl or xe2x80x94C(xe2x95x90O)alkyl. More preferably, X is O; and one of R1 and R2 is xe2x80x94(L)n-sm and the other of R1 and R2 is xe2x80x94C(xe2x95x90O)xe2x80x94R4. Preferably, L is xe2x80x94C(xe2x95x90O)xe2x80x94. Preferably, R4 is xe2x80x94N(H)alkyl or N-piperidinyl. More preferably, Z1 is xe2x80x94C(xe2x95x90O)CH3; and Z2 is dimethoxytrityl.
The support medium may be a controlled pore glass, oxalyl-controlled pore glass, silica-containing particles, polymers of polystyrene, copolymers of polystyrene, copolymers of styrene and divinylbenzene, copolymers of dimethylacrylamide and N,Nxe2x80x2-bisacryloylethylenediamine, soluble support medium or PEPS.
Z1 may be a trimethylsilyl, triethylsilyl, t-butyldimethylsilyl, t-butyldiphenylsilyl, triphenylsilyl, benzoylformyl, acetyl, chloroacetyl, dichloroacetyl, trichloroacetyl, trifluoroacetyl, pivaloyl, benzoyl, p-phenylbenzoyl, 9-fluorenylmethoxycarbonyl, levulinyl or acetoacetyl groups.
Z2 may be a 4,4xe2x80x2-dimethoxytrityl (DMT), monomethoxytrityl, 9-phenylxanthen-9-yl (Pixyl), 9-(p-methoxyphenyl)xanthen-9-yl (Mox), t-butyl, t-butoxymethyl, methoxymethyl, tetrahydropyranyl, 1-ethoxyethyl, 1-(2-chloroethoxy)ethyl, 2-trimethylsilylethyl, p-chlorophenyl, 2,4-dinitrophenyl, benzyl, 2,6-dichlorobenzyl, diphenylmethyl, p,p-dinitrobenzhydryl, p-nitrobenzyl, triphenylmethyl, trimethylsilyl, triethylsilyl, t-butyldimethylsilyl, t-butyldiphenylsilyl, triphenylsilyl, benzoylformate, acetyl, chloroacetyl, trichloroacetyl, trifluoroacetyl, pivaloyl, benzoyl, p-phenylbenzoyl, mesyl, tosyl, 4,4xe2x80x2,4xe2x80x3-tris-(benzyloxy)trityl (TBTr), 4,4xe2x80x2,4xe2x80x3-tris-(4,5-dichlorophthalimido)trityl (CPTr), 4,4xe2x80x2,4xe2x80x3-tris(levulinyloxy)trityl (TLTr); 3-(imidazolylmethyl)-4,4xe2x80x2-dimethoxytrityl (IDTr), 4-decyloxytrityl (C10Tr), 4-hexadecyloxytrityl (C16Tr), 9-(4-octadecyloxyphenyl)xanthene-9-yl (C18Px), 1,1-bis-(4-methoxyphenyl)-1xe2x80x2-pyrenyl methyl (BMPM), p-phenylazophenyloxycarbonyl (PAPoc), 9-fluorenylmethoxycarbonyl (Fmoc), 2,4-dinitrophenylethoxycarbonyl (DNPEoc), 4-(methylthiomethoxy)butyryl (MTMB), 2-(methylthiomethoxymethyl)-benzoyl (MTMT), 2-(isopropylthiomethoxymethyl)benzoyl (PTMT), 2-(2,4-dinitrobenzenesulphenyloxymethyl) benzoyl (DNBSB), or levulinyl groups.
In another embodiment, the invention is directed to a method for functionalizing a support medium with a first monomeric subunit, comprising the steps of:
providing a support bound compound of Formula I: 
xe2x80x83wherein
X is CH2, O, S or NR3;
R3 is alkyl, xe2x80x94C(xe2x95x90O)alkyl or an amino protecting group;
one of R1 and R2 is xe2x80x94(L)n-sm and the other of R1 and R2 is xe2x80x94C(xe2x95x90O)xe2x80x94R4 or xe2x80x94C(xe2x95x90S)xe2x80x94R4;
L is a linking moiety;
n is 0 or 1;
sm is a support medium;
R4 is xe2x80x94O-alkyl, xe2x80x94N(J1)J2;
J1 is H or alkyl;
J2 is alkyl or a nitrogen protecting group;
or J1 and J2 together with the nitrogen atom to which they are attached form a ring structure; and
Z1 and Z2 are orthogonal hydroxyl protecting groups;
selectively deblocking one of said orthogonal hydroxyl protecting groups to give a reactive hydroxyl group; and
treating said reactive hydroxyl group with a first monomeric subunit having an activated phosphorus group and a further protected hydroxyl group thereon for a time and under conditions sufficient to form a monomer-functionalized support medium.
In certain embodiments, the method may further comprise the steps of:
treating said monomer-functionalized support medium with a capping agent; and
optionally, treating said monomer-functionalized support medium with an oxidizing agent.
In other embodiments, the method includes the further steps of:
deblocking said further protected hydroxyl group to give a reactive hydroxyl group;
treating the reactive hydroxyl group with a further monomeric subunit having an activated phosphorus group and a further protected hydroxyl group thereon for a time and under conditions sufficient to form an extended compound;
treating said extended compound with a capping agent;
optionally, treating said extended compound with an oxidizing or sulfurizing agent;
repeating the preceding four steps one or more times to form a further extended compound; and
treating said further extended compound with an oxidizing or sulfurizing agent to form an oligomeric compound.
In certain embodiments, said last treating step cleaves said oligomeric compound from said support medium. Preferably, said last treating step is effective to remove protecting groups present on said oligomeric compound. Preferably, said cleaved oligomeric compound has a terminal hydroxyl group at the site of cleavage and, more preferably, said terminal hydroxyl group is attached to a 2xe2x80x2- or 3xe2x80x2-position of a nucleoside that is located at the 3xe2x80x2-terminus of said oligomeric compound.
In certain other embodiments, the process further comprises the step of treating said oligomeric compound with a reagent effective to cleave said oligomeric compound from said support medium. Preferably, said treating step is effective to remove protecting groups present on said oligomeric compound. Preferably, said cleaved oligomeric compound has a terminal hydroxyl group at the site of cleavage and, more preferably, said terminal hydroxyl group is attached to a 2xe2x80x2- or 3xe2x80x2-position of a nucleoside that is located at the 3xe2x80x2-terminus of said oligomeric compound.
Preferably, the treating step of said reactive hydroxyl group with a monomeric subunit having an activated phosphorus group and a further protected hydroxyl is performed in the presence of an activating agent.
Preferably, said monomeric subunit having an activated phosphorus group is a phosphoramidite, an H-phosphonate or a phosphate triester.
Preferably, said hydroxyl protecting group Z1 and each of said further hydroxyl protecting groups are acid labile.
In certain preferred embodiments of the process, said hydroxyl protecting group Z1 and each of said further hydroxyl protecting groups are removed by contacting said hydroxyl protecting groups with an acid, wherein the acid is formic acid, acetic acid, chloroacetic acid, dichloroacetic acid, trichloroacetic acid, trifluoroacetic acid, benzenesulfonic acid, toluenesulfonic acid, or phenylphosphoric acid.
Preferably, the oligomeric compounds may be oligonucleotides, modified oligonucleotides, oligonucleotide analogs, oligonucleosides, oligonucleotide mimetics, hemimers, gapmers and chimeras.